Since the discovery of the first human microRNAs (miRNAs) about a decade ago, examples of miRNA regulation have been found for virtually every cellular process (Kim et al., 2009, Krol et al., 2010). Precursors of miRNAs undergo a series of processing steps after transcription to generate an active product. In this canonical pathway, a newly transcribed primary miRNA (pri-miRNA) with at least one hairpin structure is cleaved within the nucleus by an RNAseIII enzyme, Drosha, that acts in complex with DGCR8. The resulting pre-miRNA is exported to the cytoplasm, where another RNAseIII, Dicer, removes the “terminal loop region”, or pre-element (preE), to yield the mature miRNA (FIG. 1A). Mechanisms of transcriptional control have been analyzed for many miRNAs, but the recent identification of post-transcriptional regulators of miRNA biogenesis now provides a way to investigate the molecular details of miRNA maturation and regulation (Davis-Dusenbery and Hata, 2010, Siomi and Siomi, 2010).
The let-7 family of miRNAs regulates many factors that control cell fate decisions, including oncogenes (c-Myc, Ras, HMGA-2) and cell cycle factors (CyclinD1, D2) (Bissing et al., 2008, Viswanathan and Daley, 2010). Deregulation of let-7 influences tumorigenicity of breast cancer stem cells (Yu et al., 2007a). Moreover, IL-6 is a target of let-7, thereby bridging the inflammation and cell-transformation signaling pathways (Iliopoulos et al., 2009). There are several let-7 family members in mammals, with similar mature regions but divergent sequences in the preE removed by Dicer (FIG. 1A). The preEs comprise low sequence identity and thus a minimum motif, i.e., minimal structural elements (stem, bulge, and loop), that are important for regulation of pre-miRNAs are not known (FIG. 1B).
Lin28, originally discovered as a heterochronic gene regulating developmental timing in worms (Moss et al., 1997), blocks let-7 biogenesis (Heo et al., 2008, Lehrbach et al., 2009, Newman et al., 2008, Rybak et al., 2008, Viswanathan et al., 2008). Its effects on gene expression are profound enough to make Lin28 one of the four factors sufficient to reprogram human somatic cells into induced pluripotent stem (iPS) cells (Yu et al., 2007b). Lin28 is activated in many human tumors (˜15%) and appears to be associated with less differentiated cancers (Viswanathan et al., 2009). Studies with patient samples show correlation between over-expression or mutation of Lin28 with ovarian cancer (Peng et al., 2010, Permuth-Wey et al., 2011) and colon cancer (King et al., 2011). Variations in Lin28 have also been linked to developmental traits such as height and timing of puberty onset in humans and mice (Lettre et al., 2008, Lu et al., 2009, Ong et al., 2009, Perry et al., 2009, Sulem et al., 2009, Zhu et al., 2010).
Because it is one of few specific inhibitors of miRNA maturation to be discovered thus far, understanding Lin28 activity provides an avenue for investigating the mechanisms of miRNA biogenesis and regulation. Lin28 contains two well-known nucleic acid interaction domains—a cold shock domain (CSD) and two tandem Cys-Cys-His-Cys (CCHC)-type zinc-binding motifs (CCHCx2). Mammals have two paralogs, Lin28a and Lin28b, with different physiological expression patterns but similar behavior in vitro (Guo et al., 2006, Heo et al., 2008, Viswanathan et al., 2008, Yang and Moss, 2003). Lin28 binds precursor forms of let-7 miRNAs and can inhibit both pri-let-7 processing by Drosha (Newman et al., 2008, Viswanathan et al., 2008) and pre-let-7 processing by Dicer (Heo et al., 2008, Lehrbach et al., 2009, Rybak et al., 2008). Furthermore, Lin28 can recruit a terminal uridylyl transferase (TUTase) that adds uridine to the 3′ end of pre-miRNA to increase decay (Hagan et al., 2009, Heo et al., 2009, Lehrbach et al., 2009). Although parts of the preE segment are dispensable for pri-miRNA processing by Drosha (Han et al., 2006), point mutations in the preE can disrupt interactions with Lin28 (Heo et al., 2009, Lehrbach et al., 2009, Newman et al., 2008, Piskounova et al., 2008), thereby de-repressing Drosha-mediated processing (Newman et al., 2008). Sequence variability among preEs in let-7 (FIG. 8A) has hindered interpretation of these results and extension of the conclusions to other let-7s, highlighting the need for an atomic-level view of divergent Lin28:let-7 complexes.
Accordingly, there is need in the art for inhibitors of Lin28 polypeptide activity.